90 GERM-LINE TRANSMISSION OF DONOR MITOCHONDRIAL DNA IN NUCLEAR TRANSFER-DERIVED COWS

Abstract

In embryos derived by nuclear transfer (NT), fusion, or injection of donor cells with recipient oocytes caused mitochondrial heteroplasmy. Previous studies have reported varying patterns of mitochondrial DNA (mtDNA) transmission in cloned calves. Distribution of donor mtDNA found in offspring of NT-derived founders may also vary from donor–host embryo heteroplasmy to host embryo homoplasmy. Here we examined the transmission of mtDNA from NT cows to their progeny. NT cows were originally produced by fusion of enucleated oocytes with Jersey (J) or Holstein (H1) oviduct epithelial cells, or Holstein (H2) or Japanese Black (B) cumulus cells, as previously reported (Goto et al. 1999 Anim. Sci. J. 70, 243–245; Yonai et al. 2005 J. Dairy Sci. 88, 4097–4110; Akagi et al. 2003 Mol. Reprod. Dev. 66, 264–272). Transmission of donor cell mtDNA was analyzed by PCR-mediated single-strand conformation polymorphism (PCR-SSCP) analysis of the mitochondrial D-loop region. Eleven NT founder cows were analyzed, 4 (2 = J-NT, and 2 = H1-NT) of them were heteroplasmic whereas 7 (1 = J-NT, 1 = H1-NT, 2 = H2-NT, and 3 = B-NT) were homoplasmic for the host embryo mitochondria. The proportions of donor mtDNA detected in one J-NT cow was 7.7%, and those of other cow lineages were <2%. Heteroplasmic NT cows delivered a total of 9 progeny. Four of the 9 progeny exhibited heteroplasmy with high percentages of donor cell mtDNA populations (52%, 37%, 17%, and 43%). The other 5 progeny were obtained from heteroplasmic NT cows, and all samples of the 10 progeny obtained from the homoplasmic NT cows did not harbor detectable donor cell mtDNA. A genetic bottleneck in the female germ-line will generally favor the transmission of a single mitochondrial population, leading to a return to homoplasmy. Thus, some of progeny maintained heteroplasmy with a higher ratio than that of their NT mothers, which may also reflect a segregation distortion caused by the proposed mitochondrial bottleneck. These results demonstrated that donor mtDNA in NT cows could be transmitted to progeny with varying efficiencies, in a lineage-specific fashion.